The goal of this study is to establish the importance of regulation of the activity of the pyruvate dehydrogenase complex (PDC) to glucose homeostasis in the well-fed state, fasting, and diabetes. The fate of three carbon compounds (lactate, pyruvate, alanine) is determined in large part by the relative activity of PDC. Decarboxylation of pyruvate by PDC precludes formation of glucose from three carbon compounds. In the fed state, PDC must be sufficiently active for flux into the citric acid cycle to help meet the metabolic demand for ATP. In the fasted state the complex has to be inactive to avoid loss of the compounds required for glucose synthesis. In the diabetic state, PDC is also inactive and therefore contributes to the hyperglycemia. Because of its importance for glucose homeostasis, we believe better treatment strategies for diabetes will result from a more complete understanding of the molecular mechanisms responsible for PDC regulation. Our work has helped show that PDC activity is controlled by concurrent but opposite changes in the expression of the kinases and phosphatases that set its phosphorylation state. PDC is inactivated in starvation and diabetes because of induction of greater kinase activity and less phosphatase activity. Factors that control expression of PDK4, the kinase most dramatically induced by fasting and diabetes, include insulin, glucocorticoids, and free fatty acids. We present evidence that insulin down regulates PDK4 gene transcription by activating PKB, which phosphorylates and thereby induces the release of transcription factor Foxo1 from three IREs in the PDK4 gene promoter. Glucocorticoids strongly up regulate PDK4 gene expression via the glucocorticoid receptor and a GRE in the promoter region of the human PDK4 gene. Based on these findings, our working hypothesis is that glucose homeostasis depends upon regulation of the activity state of PDC, which in turn depends upon hormonal and nutrient control of PDK and PDP expression. PDK4 knockout mice, produced to test this hypothesis, are more glucose tolerant, more insulin sensitive, and less able to maintain blood glucose levels during fasting relative to wild type mice. Our specific aims are to: (a) establish the molecular mechanisms responsible for insulin down regulation and glucocorticoid and free fatty acid up regulation of PDK4 gene expression and (b) establish the importance of regulation of PDK4 and PDK2 expression to glucose homeostasis.